1. Field of the Invention
The present invention relates to a biologically active protein having extended blood-circulation life or reduced immunogenicity and to a method of production thereof.
2. Description of the Prior Art
Biologically active proteins are expected to be used as effective drugs. Progress of gene recombination technology has recently allowed the production of such proteins on a large scale. In some cases, however, the biological activity of a protein administered to a living body remains effective only for a short time, due to its extremely rapid elimination in the circulation. In addition, administration of biologically active protein obtained from organisms other than human, such as microorganisms or animals, to human, may result in critical symptoms due to the immune reaction Therefore, technology development is desired which delays the rate of elimination of the protein from the body and which further reduces immunogenicity (antigenicity) thereof while activity is retained.
For the purpose of reducing the rate of elimination and immunogenicity, there is a method of modifying biologically active proteins with polyethylene glycol.
Polyethylene glycol itself is poorly antigenic, and when combined with an immunogenic protein, it is known to reduce the immunogenicity of the protein. Proteins modified with polyethylene glycol are said to be enhanced in blood-circulation life, and thus to maintain biological activity for a longer time. Means available for coupling polyethylene glycol to a protein include a method using polyethylene glycol methylether and cyanuric chloride (or fluoride) and a method using carboxyl derivatives of polyethylene glycol. As the former requires a relatively high, alkaline pH at the reaction, it cannot be applied to biologically active proteins which will be inactivated under alkaline conditions. Moreover, the toxicity of cyanuric chloride itself is troublesome. In the latter, coupling agents such as carbodiimide may cause inter- or intra-molecular crosslinking of the protein, and, furthermore, the destruction of the active conformation of protein may occur due to neutralization of the positively charged groups on the protein by the reaction. In another method, where either alkyl- or alkanoyl-polyethylene glycol aldehyde is introduced into biologically active protein in the presence of a boronic reducing agent, the reducing agent may break the disulfide bond related to the maintenance of the active conformation of protein, and may decrease the biological activity of the protein and insolubilize the protein by changing higher order structure.